An improved process for preparation of S-Acetyl-l-glutathione

Article abstract of DOI:10.1021/acs.oprd.5b00081

An efficient one-step synthesis of S-acetyl-l-glutathione has been developed in a DMF-TFA mixed solvent with CoCl₂ as catalyst. This process not only has the advantages of selective acylation of the glutathione thiol group without involving the free amino but also highlights recycling of the relatively costly solvent TFA, which improves the yield to 91% and quality to 99.7%. The reaction is cost-effective, efficient, and easy to scale-up.

Full text of DOI:10.1021/acs.oprd.5b00081

Technical Note
pubs.acs.org/OPRD
An Improved Process for Preparation of S‑Acetyl‑L‑glutathione
Kai Fu, Qiu-Fen Wang, Fu-Xu Zhan, Liu Yang, Qian Yang, and Geng-Xiu Zheng*
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, Shandong, P.R. China
S
* Supporting Information
ABSTRACT: An efficient one-step synthesis of S-acetyl-L-glutathione has been developed in a DMF−TFA mixed solvent with
CoCl2 as catalyst. This process not only has the advantages of selective acylation of the glutathione thiol group without involving
the free amino but also highlights recycling of the relatively costly solvent TFA, which improves the yield to 91% and quality to
99.7%. The reaction is cost-effective, efficient, and easy to scale-up.
of glutathione without involving the free amino group and the
adoption of low cost and easily available reagents attracted us to
this strategy. Nevertheless, this process would result in poor
quality and difficult recycling of the relatively expensive solvent
trifluoroacetic acid, owing to the water added to the reaction
mixture.
Herein we decided to overcome these difficulties and control
the quality of 1 by modifying the method and effectively
optimizing the reaction parameters.
INTRODUCTION
■
Glutathione (γ-glutamyl-cysteinyl-glycine, GSH), a thiol group-
containing tripeptide, serves as an important intracellular water-
soluble antioxidant and detoxifying agent.1 Recently, gluta-
thione has attracted an increasing interest due to the potential
therapeutic effects on cancer and AIDS.2 However, GSH
cannot be directly absorbed by cells; it needs to be broken
down into amino acids and resynthesized to GSH in cells, and
its half-life is very short in serum.3 In addition, the synthesis
process of GSH is often damaged by viral infections. So
replenishment of intracellular GSH is still a challenging goal.
These obstacles could be overcome by transforming glutathione
to its derivatives.4
Previous studies have demonstrated that supplementation
with a glutathione pro-drug, such as N-acetylcysteine (NAC),
S-acetylcysteine (SAC), N-acetylglutathione (N-GSH), and S-
acetylglutathione (S-GSH), is more efficient than GSH for
intracellular GSH supplementation.5 However, N-acetyl de-
rivatives may have neurotoxic effects on the nerves for patients
with glutathione synthetase deficiency.6 S-Acetyl derivatives,
such as S-acetyl-L-glutathione (S-GSH), are promising agents
for GSH restoration, which is not only more stable in plasma
but also taken up directly by cells and then converted into GSH
by intracellular thioesterases.7
Quite a few methods for selective acylation of the thiol group
of glutathione without involving the free amino group have
been reported (Scheme 1). However, these methods have
various drawbacks and are not suitable for large-scale
applications. For example, the initial method involving
exchange reaction of acetyl coenzyme A with glutathione
(Scheme 1a) is unsuccessful in isolating product from the
reaction mixture.7 Subsequently, Wieland and Bokelmann9 have
synthesized S-GSH using the exchange reaction between S-
acetylthiophenol and glutathione in 70% yield (Scheme 1b).
This method needs at least 10-fold molar S-acetylthiophenol
under enzyme catalysis. Kielley and co-workers10 replace S-
acetylthiophenol with cheap thioacetic acid to prepare S-GSH
in 40% yield. All of the above-mentioned enzymatic processes
are not suitable for industrial scale preparation of S-GSH at
present, because of the dependence on enzyme and tedious
post-treatment. The alternative approach for the synthesis of S-
GSH, reported by Galzigna,11 was found to be promising for
scale-up (Scheme 1c). The selective acylation of the thiol group
RESULTS AND DISCUSSION
■
In the method reported by Galzigna, byproduct 3 was formed
in the reaction (up to 0.78 area %, HPLC) and difficult to be
removed during purification, which affected the stability and
safety of the S-GSH. Therefore, controlling and minimizing the
impurity are the key issues in the production of S-GSH.12
It is reported that N-acylglycine derivatives can be induced to
undergo intramolecular dehydration in the presence of a
dehydrating agent such as N,N-dicyclocarbodiimide (DCC) at
0 °C for 2 h or acetic anhydride refluxing 30 min.13 We
attempted to find an efficient catalyst to improve the reaction
rate and selectivity and to reduce the byproduct 3.
It is noted that Zn, ZnO,14 CoCl2,15 and BiCl3 are active
16
catalysts for the synthesis of some thiol esters with acetyl
chloride at room temperature. A large number of experiments
were carried out to screen an effective catalyst for the acylation
of glutathione with acetyl chloride, and the results are presented
in Table 1.
We added acetyl chloride at 0−5 °C in 5 min and performed
the reaction at room temperature (20−30 °C). As shown in
Table 1, although Zn and ZnO are active catalysts for the
acylation of some thiols with acetyl chloride at room
temperature, ZnCl2 is not effective in catalyzing acylation of
GSH with acetyl chloride (Table 1, entry 2−4). The results
indicate that CoCl2 and BiCl3 are very active for the acylation
reaction and CoCl2 is more effective than BiCl3. The best result
was obtained on treatment of GSH with acetyl chloride in the
presence of 0.5% mol CoCl2. The yield of S-GSH was 89% in
10 min (Table 1, entry 6). In this case, the impurity content
was minimal (0.02%, assay by HPLC).
Received: January 15, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.oprd.5b00081
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Technical Note
Scheme 1. Reported synthetic routes of S-GSH
a
a
Table 1. Effect of catalyst on the acylation reaction
Table 2. Effect of the amount of acetyl chloride used
d
b
b
c
molar (%
mol)
time
conversion
bc
1
3
(area
%)
entry
molar (equiv)
time (min)
yield (%)
purity (%)
,
entry catalyst
(min)
(%)
100
(%)
1
2
3
4
5
1.00
1.05
1.10
1.15
1.20
10
10
10
10
10
78
81
89
89
89
99.4
99.6
99.7
99.5
99.5
1
2
none
40
30
20
20
20
10
5
80
81
76
77
87
89
88
87
86
82
0.78
0.47
0.42
0.49
0.03
0.02
0.03
0.07
0.07
0.06
ZnCl2
ZnCl2
ZnCl2
CoCl2
CoCl2
CoCl2
BiCl3
BiCl3
BiCl3
1
5
100
95
3
4
10
0.1
0.5
2
96
5
97
a
Reaction conditions: glutathione (10.0 g, 0.0325 mol), TFA (80 mL),
CoCl2 0.5% mol, addition temperature 0−5 °C, reaction temperature
20−30 °C. Isolated yield. Detected by HPLC.
6
100
98
b
c
7
8
0.1
0.5
2
20
10
5
100
100
95
9
the amount of acetyl chloride showed no difference in the yield
of compound 1.
10
a
Reaction conditions: glutathione (10.0 g, 0.0325 mol), TFA (80 mL),
Trifluoroacetic acid is a relatively expensive reagent. Before
proceeding with large-scale synthetic efforts, we turned
attention to select a mixed solvent and an appropriate volume
ratio. Thus, we screened various solvents to identify the
conditions that would lead to good results. The results are
summarized in Table 3.
b
c
acetyl chloride (5.1 g, 0.065 mol). Detected by HPLC. Conversion
d
was calculated from the HPLC area. Isolated yield.
The plausible reaction mechanism for the acetylation of GSH
with acetyl chloride using cobalt(II) chloride as catalyst at room
temperature is shown in Figure 1.17
As shown in Table 3, when the volume ratio of DCM to TFA
was increased from 1:4 (v/v) to 1:2 (v/v), the yield and purity
were not obviously decreased. The mixed solvent of DMF/TFA
exhibited even better results until the DMF load up to 50%.
However, the reactions proceeded in a mixed solvent of
AcOH/TFA (1/2, v/v) or toluene/TFA (1/4, v/v) with only
60% and 40% yields, respectively, and purity was also not
satisfactory. We suspected it might be that the trifluoroacetic
salt of GSH was soluble in polar aprotic solvents, leading to the
reaction proceeding further towards the formation of desired
product. Unfortunately, this salt was insoluble in mixed solvent
of AcOH/TFA or toluene/TFA, which led to poor yields.
Although the mixed solvent of TFA/DCM showed satisfactory
results, it was challenging to separate TFA from the recovered
solvent in subsequent experiments. For this reason, TFA/DMF
(1/1, v/v) was chosen as the ideal reaction mixed solvent rather
than TFA/DCM.
Figure 1. Plausible reaction mechanism for the acetylation reaction
with CoCl2 catalyst.
The next focus of our study was to optimize the molar
equivalents of the acetyl chloride. We carried out this reaction
by using varying amounts of acetyl chloride, and the results are
summarized in Table 2. It was found that the yield of
compound 1 increased with an increase in amount of acetyl
chloride up to 1.1 equiv. At 1.1 equiv, compound 1 was
obtained in 89% yield (Table 2, entry 3). A further increase in
B
DOI: 10.1021/acs.oprd.5b00081
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Technical Note
be separated due to the similar boiling points. So, we chose
methanol as the quencher.
Table 3. Results of acylation reaction carried out in different
solvents
a
With optimized reaction conditions as described above (1.1
equiv of acetyl chloride, addition temperature of 0−5 °C,
reaction temperature of 20−30 °C, solvent of TFA/DMF (1:1,
v/v), reaction time of 10 min, and quencher of methanol), a
few reactions were executed on pilot scale. When completed,
the reaction mixture was subjected to vacuum fractional
distillation. Fraction 1 mainly contained the unreacted
methanol and TFA. Fraction 2 mainly contained DMF.
Acetyl chloride was added dropwise to Fraction 1 at room
temperature. After stirring for 1 h at room temperature,
atmospheric distillation process was used to purify TFA from
Fraction 1. We removed the front fraction (<70 °C) and
collected the fraction of 70−75 °C. The collected TFA was
analyzed by GC for quality (Table 5). As shown in Table 5, we
first attempted to add 1 equiv of acetyl chloride to remove the
methanol (Table 5 entry 1). In this case, the purity of TFA was
only 97.6%. Then by increasing the amount of acetyl chloride
to 2 equiv, we were able to achieve 99.2% purity with the
residue of only <0.1% methanol (entry 3). Three experiments
were performed using the collected TFA. The results showed
that the yield of S-GSH was 89%, 90%, and 90%, respectively.
b
c
entry
solvent
quantity (v/v)
yield (%)
purity (%)
1
2
DCM/TFA
DCM/TFA
DCM/TFA
DCM/TFA
AcOH/TFA
AcOH/TFA
AcOH/TFA
toluene/TFA
toluene/TFA
DMF/TFA
DMF/TFA
DMF/TFA
DMF/TFA
DMF/TFA
1/4
1/3
1/2
1/1
1/4
1/3
1/2
1/5
1/4
1/4
1/3
1/2
1/1
2/1
88
99.6
99.5
99.5
99.4
99.3
99.0
98.5
95.2
86.7
99.7
99.6
99.6
99.7
98.5
87.8
87.6
87.1
87.2
80.0
60.0
62
3
4
5
6
7
8
9
40
10
11
12
13
14
90.5
91.0
90.8
91.0
88.4
a
Reaction conditions: glutathione (10.0 g, 0.0325 mol), solvent (80
mL), acetyl chloride (2.8 g, 0.036 mol), CoCl2 0.5% mol, addition
temperature 0−5 °C, reaction temperature 20−30 °C. Isolated yield.
b
c
Detected by HPLC.
As discussed above, we should resolve the issue of recycling
of the relatively expensive solvent trifluoroacetic acid. Hence, it
became important for us to recover this solvent and establish a
recycling procedure in subsequent experiments. First we
quenched the reaction with water following Galzigan’s work,
and the TFA was recycled by atmospheric distillation. However,
the recycled TFA was not able to be reused, because the
residual water in trifluoroacetic acid was not able to be removed
by atmospheric distillation, which would affect the reaction
process. Then P2O5 was added to the recycled TFA but failed
to remove the water. To avoid the water, we decided to use
alcohol as the quencher, and excess alcohol was transformed to
acetate by addition of acetyl chloride before atmospheric
distillation. On the basis of the experimental results, we
discovered both methanol and ethanol gave promising results,
which are summarized in Table 4. Methyl acetate was easily
CONCLUSIONS
■
An improved process for the preparation of S-acetyl-L-
glutathione (1) was developed and optimized. It was found
that CoCl2 is an effective catalyst for the acylation of GSH by
acetyl chloride in the mixed solvent of TFA/DMF (1:1, v/v) at
room temperature. Recycling of solvent TFA could further
increase the economic benefits. The current process increased
the overall productivity by 6%.
EXPERIMENTAL SECTION
General. Glutathione and other reagents and solvents were
■
obtained from commercial suppliers and used without further
1
purification. H and 13C NMR spectra were recorded on a
Bruker 400 MHz spectrometer in DMSO-d6; chemical shift
data were reported in δ (ppm) from the internal standard TMS.
The IR spectra were recorded on KBr pellets on a PerkinElmer
FTIR (spectrum one) spectrophotometer. The reaction
monitoring and purity (area percentage) were analyzed by
high-performance liquid chromatography (HPLC) at λ = 210
nm using Inertsil NH2 column (250 mm × 4.6 mm, 5 μm) at
1.0 mL/min flow. The purity of TFA was analyzed by gas
chromatography Agilent 7890A and a HP-FFAP capillary
column (capillary dimension = 30 m × 320 μm × 0.25 μm),
oven temperature = 60−200 °C, ramp = 15 °C/min, detector
heater = 250 °C; N2 flow = 25 mL/min, H2 flow = 30 mL/min,
and air flow = 400 mL/min.
a
Table 4. Effect of quencher on the quenching reaction
temp.
(°C)
time
yield
b
purity
c
entry quencher equiv
(min)
(%)
(%)
1
2
3
water
4
4
4
Rt
Rt
Rt
20
20
20
90
91
90
99.6
99.5
99.6
methanol
ethanol
a
Reaction conditions: glutathione (10.0 g, 0.0325 mol), solvent (TFA
40 mL and DMF 40 mL), CoCl2 0.5% mol, acetyl chloride (2.8 g,
0.036 mol), addition temperature 0−5 °C, reaction temperature 20−
b
c
30 °C. Isolated yield. Detected by HPLC.
Preparation of S-GSH. Acetyl chloride (1.4 kg, 17.83 mol)
was added dropwise to a solution of L-glutathione (5.0 kg, 16.27
mol) in the presence of trifluoroacetic acid (15 L), DMF (15
separated from the recycled TFA because of the big gap
between their boiling points, while ethyl acetate was difficult to
a
Table 5. Recovery and recycling of TFA from fraction 1
c
content (%)
a
b
entry
fraction 1 (g)
acetyl chloride equiv
temp. (°C)
CH3OH
methyl acetate
quality of TFA
quantity of TFA (g)
1
2
3
700
700
700
1
70−75
70−75
70−75
<1.2
<0.8
<0.1
<0.4
<0.4
<0.1
>97.6
>98.6
>99.2
602
611
623
1.5
2
a
b
c
Acetyl chloride molar equiv. Collection temperature. Determined by GC.
C
DOI: 10.1021/acs.oprd.5b00081
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Technical Note
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L), and CoCl2 (10.56 g, 0.081 mol) at 0−5 °C over a period of
5 min. Thereafter, the temperature was raised to 20−30 °C, and
the reaction mixture was stirred for 10 min. The progress of the
reaction was monitored by HPLC. After completion, the
reaction was quenched by addition of 0.21 L of methanol, and
the reaction mixture was stirred for an additional 20 min at
room temperature. The TFA and DMF were removed in vacuo,
and ethyl acetate (50 L) was added to the resulting oil. The
mixture was cooled to 0 °C obtaining a white solid overnight.
The crude precipitate was dissolved in 20 L of warm water (40
°C). The obtained solution was further diluted with 60 L of
acetone. After cooling in an ice−water bath for about 2 h, the
white crystalline material formed was collected, washed with
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vacuum affording S-acetyl-L-glutathione (5.20 kg, yield 91.5%).
Chromatographic purity: 99.7% (by HPLC). mp 202−203
°C. [α]D20 = −16.5 (1.0 water). 1H NMR (400 Hz, DMSO-d6):
δ (ppm) 8.71 (t, J = 6.0, 1H), 8.47 (d, J = 8.8, 1H), 4.39 (m,
1H), 3.68 (d, J = 6.0, 2H), 3.36 (m, 2H), 3.28 (t, J = 6.4, 2H),
2.93 (m, 1H), 2.31 (s, 3H), 2.28 (m, 2H), 1.90 (m, 1H), 1.83
(m, 1H). 13C NMR (400 Hz, DMSO-d6): δ (ppm) 196.38,
172.24, 171.35, 170.74, 170.63, 53.48, 52.33, 41.69, 31.82,
31.00, 30.93, 27.21. IR (KBr, cm−1): 3355, 3059, 2956, 2729,
1701, 1677, 2648, 1514, 1432, 1353, 1231, 1132, 963, 636.
Recycling Process for TFA. Acetyl chloride (1.7 kg) was
slowly added to fraction 1 (7 kg) at room temperature.
Thereafter, the reaction mixture was stirred for 1 h at room
temperature. An atmospheric distillation process was used to
separate TFA from the Fraction 1. We removed the front cut
fraction (<70 °C) and collected the fraction of 70−75 °C (6.3
kg, purity 99.2%, area %).
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, IR spectrum, and HPLC chromatogram of S-acetyl-L-
glutathione. GC chromatogram of recovered TFA. The
Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.oprd.5b00081.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: chm_zhenggx@ujn.edu.cn. Tel.: +8653182765841.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our college at foundation and analysis for their
contributions to the project.
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DOI: 10.1021/acs.oprd.5b00081
Org. Process Res. Dev. XXXX, XXX, XXX−XXX